Iron Nutritional Status And Serum Metabolomics Analysis Of Female Patients With Nonalcoholic Fatty Liver Disease In Qingdao

Objective:In this study,the relationship between iron metabolism and female nonalcoholic fatty liver disease(NAFLD)was analyzed from the aspects of dietary iron intake and serum iron metabolism indexes.New biomarkers of NAFLD were explored by serum metabonomics analysis,and the correlation between new biomarkers and indexes of liver and kidney function,glucolipid metabolism and iron metabolism was analyzed,so as to provide a theoretical basis for guiding women to prevent NAFLD reasonably and improve NAFLD through diet.Methods:1.A case-control study design was adopted.From July to December 2020,136 female NAFLD patients in a Qingdao community were recruited as the case group,and135 healthy adult women were recruited as the control group.Basic information and data of dietary intakes were collected through questionnaires.Fasting venous blood was collected for the detection of various laboratory indicators: blood routine indicators were determined by hematology analyzer;blood biochemical indexes were determined by automatic biochemical analyzer;serum insulin and ferritin levels were detected by radioimmunoassay;the levels of serum iron,zinc and selenium were detected by inductively coupled plasma mass spectrometry;serum hepcidin and hemojuvelin(HJV)levels were determined by enzyme-linked immunosorbent assay.SPSS 24.0 software was used for statistical analysis of data.T test(normal distribution data)or Mann-Whitney U test(skewness distribution data)was used for measurement data,and Chi-square test was used for categorical data.Multivariate logistic regression analysis was used to explore the relationship between levels of serum nutrients and daily intakes of dietary nutrients and NAFLD.Graph Pad Prism 8.0 software was used for drawing.2.Serum metabolomics analysis: serum samples from 20 pairs of patients and controls matched by age and gender were analyzed by technology of ultra performance liquid chromatography/high resolution mass spectrometry.Online data analysis software Metaboanalyst was used for univariate and multivariate statistical analysis: T test was used to compare the differences of metabolites between the two groups;supervised orthogonal partial least squares discriminant analysis(OPLS-DA)was used to distinguish cases and controls.SIMCA 14.1 software was used to calculate the Variable Importance for the Projection(VIP),and S-plot analysis was performed.VIP,results of S-Plot analysis and P values(VIP≥1、|P|≥0.05、|P(corr)|≥0.5、P<0.05)were considered together to find differential metabolites.Then,differential metabolites were identified by Human Metabolome Database(HMDB).Spearman rank correlation was used to analyze the correlation between differential metabolites and indexes of blood glucose,blood lipids,liver and kidney function and iron metabolism.Hem I 1.0 software was used for drawing.Results:1.Comparative analysis of basic characteristics of subjects in the two groups showed that,compared with non-NAFLD group,NAFLD patients were older and had higher BMI,systolic blood pressure,diastolic blood pressure,waist circumference,abdominal circumference and hip circumference,and the proportions of patients with diabetes,hypertension,hyperlipidemia and hyperuricemia were higher in NAFLD patients(all P values<0.05).Comparative analysis of blood routine and biochemical parameters between two groups showed that,compared with the non-NAFLD group,the levels of red blood cell count,platelet count,white blood cell count,lymphocyte count,alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,glutamyl transpeptidase,fasting blood glucose,insulin,homeostasis model assessment of insulin resistance,triglyceride,total cholesterol,low density lipoprotein cholesterol and uric acid were higher in NAFLD group,while the level of high density lipoprotein cholesterol was lower in NAFLD group(all P values<0.05).2.Comparative analysis of serum mineral and ferritin levels in the two groups showed that,compared with the non-NAFLD group,the levels of serum iron and ferritin were higher in NAFLD group,while the level of serum zinc was lower in NAFLD group(all P values<0.05).Multivariate logistic regression analysis showed that after adjusting for confounding factors,such as age,BMI,hypertension,diabetes,hyperlipidemia and hyperuricemia,there was a significant positive correlation between serum ferritin level and NAFLD,a significant negative correlation between serum zinc level and NAFLD,and no significant correlation between serum iron level and NAFLD,and the OR(95%CI)were 1.020(1.009,1.031),0.755(0.618,0.921)and 1.006(0.988,1.025),respectively.In addition,compared with the non-NAFLD group,NAFLD patients had lower serum HJV level(P value<0.05),and no significant difference was found in serum hepcidin level between the two groups(P value>0.05).3.Compared with the non-NAFLD group,NAFLD patients had lower daily intakes of crab shrimp and green leafy vegetables,and lower daily intakes of vitamin A,vitamin E,calcium,total iron and non-heme iron(all P values<0.05).Multivariate logistic regression analysis of the relationship between dietary nutrients intakes and NAFLD showed that the daily intakes of vitamin A,total iron and non-heme iron were negatively correlated with the risk of NAFLD,and the OR(95%CI)were 0.999(0.998,1.000),0.896(0.815,0.984)and 0.906(0.829,0.991),respectively.4.Serum metabolomics analysis: 6 differential metabolites identified by HMDB were found finally,which were hercynine,N-Glycolylneuraminic acid,L-ornithine,beta-Sitosterol,N-Carbamoylputrescine and behenic acid.Compared with the non-NAFLD group,the serum levels of hercynine,L-ornithine,beta-Sitosterol and N-Carbamoylputrescine were lower in NAFLD group,while the serum levels of N-Glycolylneuraminic acid and behenic acid were higher in NAFLD group.Correlation analysis showed that indexes of liver function,blood lipid and uric acid were negatively correlated with hercynine,L-ornithine,beta-Sitosterol and N-Carbamoylputrescine,while positively correlated with N-Glycolylneuraminic acid and behenic acid(all P values<0.05);Significant negative correlations were found between serum iron and beta-Sitosterol,between serum ferritin and L-ornithine,N-Carbamoylputrescine,while significant positive correlations were found between serum iron,ferritin and N-Glycolylneuraminic acid,behenic acid(all P values<0.05).Conclusion:1.Iron metabolism disorder exists in female NAFLD patients with increased serum iron and ferritin levels and decreased serum HJV level,and serum ferritin level is positively correlated with the risk of NAFLD in women.2.Dietary total iron and non-heme iron intakes were negatively correlated with the risk of NAFLD in women.3.Female NAFLD patients had decreased levels of serum hercynine,L-ornithine,beta-Sitosterol and N-Carbamoylputrescine,and increased levels of serum N-Glycolylneuraminic acid and behenic acid,and they were significantly correlated with indexes of liver enzymes,blood lipids,uric acid and iron metabolism.